Ischemic stroke is a leading cause of human death and disability. In addition to neuroprotective strategies that have failed previous clinical trials, regenerative therapies have gain escalating attention for brain repair and functional recovery after stroke. Recently, a breakthrough discovery demonstrates that transduction of non-neuronal cells such as reactive astrocytes with the panneuronal transcription factor NeuroD1 (ND1) and a few others can reprogram these cells directly into neural progenitors or even mature and functional neurons via a process called direct reprogramming/conversion that bypasses stem cell stage. Lentiviral vector delivery of ND1 to reactive astrocytes results in permanently reprogrammed functional neurons without the need for maintained ectopic expression of the gene. Thus, intra-lineage direct reprogramming implicates an unprecedented resource of endogenous neurogenesis by leveraging existing proliferative astrocytes. The proposal is a novel application of the direct reprogramming after ischemic stroke and explores its application in aged mice. This approach takes the advantages of injury-induced astrocyte activation and accumulation in the peri-infarct region. Reprogrammed new neurons, termed induced neurons (iNeurons or iNs), at the injury site are autologous and post-mitotic, which eliminate the risk of rejection and tumorigenesis of transplanted exogenous cells. In our preliminary experiments, we successfully converted astrocytes into mature neurons in vitro and in focal ischemic stroke models of the mouse. Many converted iNs were identified in the brain even one months after stroke and the conversion. Based on our in vitro and in vivo data and emerging evidence from other groups, we propose to test this regenerative therapy in a focal ischemic stroke model of aged mouse. Specific Aim 1 will study the in vitro and in vivo reprogramming of astrocytes into iNs under hypoxic/ischemic conditions. Using ND1 lentivirus packaged with the GFAP promoter and mCherry marker, we will validate the efficacy, efficiency and time windows of reprogramming reactive astrocytes as an endogenous neuronal supply for brain repair. Specific Aim 2 will test the hypothesis that direct reprogramming at the right time can reduce the physical and chemical barriers for neurogenesis. The mechanism of the benefits and a balanced microenvironment that is neuroprotective as well as permissive for regeneration will be tested. Specific 3 will study the direct conversion combined with a rehabilitative strategy of increased peripheral activities in aged mice, designed to overcome impaired neuroregeneration and neural plasticity in the aged brain. We hypothesize that the combinatorial approach promotes activity-dependent neural plasticity, circuitry repair, and functional recovery after stroke. These three Aims target coordinated but distinct regenerative mechanisms, endorsed by compelling evidence and state-of-the-art technologies. We predict that each Aim alone and/or together will provide novel strategies for a regenerative therapy.